Synthetic particulate vectors and preparation process

ABSTRACT

A synthetic particulate vector comprising a non-liquid hydrophilic nucleus which does not have an external lipid layer grafted thereon. A method for preparing a particulate vector by encapsulating an ionizable active principle, vectors obtainable through said method, and pharmaceutical, cosmetological or food compositions containing such vectors are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. applicationSer. No. 08/513,853, filed May 1, 1996. Application Ser. No. 08/513,853is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to new types of particles which canbe used alone or as vectors for various compounds. It also relates to aprocess for the preparation of particulate vectors which makes possibleimproved control of the active principle charging.

[0003] Supramolecular Biovectors or SMBV are particles which arebiomimetic of the endogenous vectors of the body and which are capableof encapsulating and of carrying a large number of active principlesfor, in particular, pharmaceutical, cosmetic or agribusiness use.

[0004] A first type of SMBV was described in Application EP 344,040.Their structure is very well suited to the role of vector, in particularas a result of the possibility of modifying their size and theircomposition according to the molecule or molecules transported and theiruse.

[0005] SMBV are synthesized in three successive steps: synthesis of acentral core composed, for example, of crosslinked naturalpolysaccharide, which can be derived by ionic groups and brought, inparticular by ultramilling, to the desired size (between 10 nanometersand a few microns, according to the desired use) establishment of a ringof fatty acids grafted covalently solely at the periphery of the centralcore, in order to confer a peripheral hydrophobic nature on the latterwhile retaining its internal hydrophilic nature stabilization of one orof a number of external lipid lamellae, composed in particular ofphospholipids or of ceramides, sometimes with the addition of otherconstituents, for example of constituents of biological membranes.

[0006] The active principles, according to their physicochemicalcharacteristics, can be transported either in the external lipidlamellae (in the case of lipophilic or amphiphilic compounds) or withinthe hydrophilic core (in the case of polar compounds).

[0007] Encapsulation of active principles of polar nature can takeplace, according to the structure of the latter, either before formationof the fatty acid ring or between this step and stabilization of theexternal lamella.

[0008] Despite their suitability for many uses, the synthesis of SMBVscan sometimes cause problems and in particular:

[0009] it requires a step which is problematic to control in graftingthe fatty acid ring;

[0010] this grafting, carried out solely at the periphery of the core,must be carried out homogeneously, which requires in particular a priordrying step, under very specific conditions;

[0011] if the active principle is encapsulated before the grafting ofthe fatty acid ring, some of these molecules, localized, after theirencapsulation, at the periphery of the core, can be derived by the fattyacid, leading to modification of the properties of this activeprinciple;

[0012] if the active principle is encapsulated after the grafting of thefatty acid ring, the latter can be detrimental to the penetration of theactive principle into the hydrophilic core.

BRIEF SUMMARY OF THE INVENTION

[0013] The present inventors have shown that, surprisingly, in certainapplications, it was possible to scale down the reaction scheme by notgrafting the ring of fatty acids and phospholipids to the periphery ofthe crosslinked hydrophilic core.

[0014] The present inventors have shown that the polysaccharideparticles thus obtained could be used as is. They are then named PS-typeSMBV, by analogy with supramolecular Biovector or PSC (polysaccharidiccore).

[0015] The present inventors have indeed shown that the polysaccharideparticles, even of small size, could be used provided that suitablecharging protocols are adopted.

[0016] This is why the subject of the present invention is a syntheticparticulate vector, characterized in that it comprises:

[0017] a non-liquid hydrophilic core.

[0018] A further subject of the present invention is a syntheticparticulate vector which consists essentially of a non-liquidhydrophilic core.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The notion of vector must, in this instance, be understood withinthe broad meaning, that is to say that it comprises particles having asupport role, for example when they are incorporated in a composition,either as such or for the transportation, the presentation and/or thestabilization of active compounds.

[0020] A non-liquid hydrophilic core (or matrix) can be a hydrophilicpolymer.

[0021] The hydrophilic matrix can in particular be composed ofpolysaccharides or oligosaccharides which are naturally or chemicallycrosslinked. The polysaccharide is preferably chosen from dextran,starch, cellulose and their derivatives.

[0022] The hydrophilic core can be obtained by various methods and inparticular, if it is a core of polysaccharide nature, by using abranched or linear biodegradable polysaccharide. This polysaccharide canbe, for example, starch or one of its derivatives. Crosslinkingprocesses are known to a person skilled in the art and can be carriedout by means of bi- or tri-functional agents, such as epichlorohydrin orphosphorus oxychloride.

[0023] The properties of the polysaccharide can be modified by graftingthe sugars by acidic or basic ionic functional groups which areimportant for the encapsulation of ionic active principles.

[0024] Encapsulation of the hydrophilic active principles can be carriedout at this stage of the synthesis. The gel obtained during thesynthetic step is then washed and partially dehydrated by means, forexample, of centrifugation techniques, then brought into the presence ofthe active principle and slowly rehydrated. As the gel has the abilityto swell with water, the active principle is carried within thepolysaccharide network where it can be bound by ionic bonds with thegroups grafted within the gel.

[0025] The gel obtained, whether it contains or does not contain anencapsulated compound, must be mechanically ground for the purpose ofobtaining particles of desired size. The ultramilling methods are knownin the state of the art and can in particular involve a high pressureextrusion using a homogenizer.

[0026] Another subject of the present invention is a process for thepreparation of a particulate vector, comprising:

[0027] a) encapsulating a basic ionizable active principle in acrosslinked hydrophilic matrix grafted by acidic ionic ligands, at a pHbelow the pK_(a) of the active principle; and

[0028] b) increasing the pH of the medium to a value above the pK_(a) ofthe active principle.

[0029] In fact, the adoption of a suitable protocol for the charging ofhydrophilic cores makes it possible to control the topology of thecharging.

[0030] The hydrophilic matrix is preferably composed of polysaccharidesor of oligosaccharides, which are naturally or chemically crosslinked.

[0031] This process, which can be used with SMBV, is more particularlyimportant with particles in which the external lipid lamellae have beenreduced (L-type SMBV) or eliminated (PS-type SMBV) with respect to themethod described above. The present inventors have observed that it isdifficult to use such SMBV containing reduced lipid lamellae as vectorsfor the encapsulation of ionic active principles with conventionalcharging methods.

[0032] In fact, if molecules of the active principle are bound with thepolysaccharide particle of the core while being maintained at theperiphery of the core, this can result in an instability in the particlesuspension, it being possible for the particles to aggregate with oneanother by virtue of interparticulate bonds due to the active principle.This phenomenon is relatively minor for low levels of charging of activeprinciples, whereas it becomes very important with high levels ofcharging of active principles. Likewise, the size of the particles isextremely important. With particles of large size (for example, greaterthan 100 manometers), the ratio of the surface area to the internalvolume of the particle is very low; for this reason, in comparison withthe total amount of active principle encapsulated, the amount of activeprinciple bound at the periphery of the particles is very low, thuslimiting the possibilities of interparticulate bonds. In contrast, whenthe particles are very small in size, this aggregation phenomenon isvery noticeable. It should also be noted that this phenomenon is notvery marked with SMBV having a layer of fatty acids grafted onto thecore, which then serves to isolate from interparticulate interactions.

[0033] In order to overcome this, the present inventors have shown thatit is possible to control topologically the penetration of the activeprinciple within the particles by controlling the ionic conditions ofthe encapsulation.

[0034] The polysaccharide cores can be regarded as polyelectrolytematrices and, as such, they have a pH differential between the insideand the outside of the particle. This phenomenon is due to the more orless significant dissociation of the counterions and to theimmobilization of the ionic functional groups on the polysaccharidenetwork. This property makes it possible to control the localization ofthe active principle to be incorporated, by causing a solely internalencapsulation or an encapsulation solely at the surface or alternativelyin the periphery of the core.

[0035] When the active principle to be incorporated is basic in nature,its solubilization in a medium with a pH below its pKa leads it to existin the ionized form; it can then become attached to the anionic groupsgrafted onto the polysaccharide core. When the pH rises above the pKa,the active principle is in a deionized form, which does not allow it tointeract with the matrix. In order to control the localization of theencapsulated active principle, use is therefore made of the pHdifferential which exists between the inside and the outside of theparticle: if the external medium has an excessively high pH, the activeprinciple cannot interact with the ionic groups placed at the peripheryof the cores. The internal pH of the cores derived by acidic ligandsbeing lower than the external pH, the active principle, which hasentered the particle in the deionized form, becomes ionic again and thusis bound to the anionic groups of the L-type SMBV. In this specificcase, the active principle will be localized solely in the core of theparticle, to the exclusion of the peripheral region. This type ofencapsulation is thus very favorable to an optimum dispersion of theparticles.

[0036] In the case of acidic active principles, it is possiblesymmetrically to apply the process with cores derived by basic ligands,according to the following steps:

[0037] a) encapsulating an acidic ionizable active principle in acrosslinked hydrophilic matrix grafted by basic ionic ligands, at a pHabove the pK_(a) of the active principle; and

[0038] b) decreasing the pH of the medium to a value below the pK_(a) ofthe active principle.

[0039] This type of charging with topological control of thelocalization of the active principle in the polysaccharide core isparticularly advantageous for vectorization applications with SMBV inwhich the external lipid lamellae have been reduced (L-type SMBV) oreliminated (PS-type SMBV) but it is also suitable and desirable for SMBValready described in the above patents, in order to increase the degreeof charging or to minimize the disturbances caused to the structure ofthe external phospholipid lamella in the case of external attachment ofmacromolecules, for example of recognition units and in particular of anapoprotein.

[0040] Another subject of the invention is a particulate vector composedof a crosslinked hydrophilic core grafted by ionic groups and herecalled PS-type SMBV. The ionic groups can be anionic groups, such as forexample phosphates, succinates or carboxymethylates, or cationic groups,for example quaternary ammoniums or amines. The size of the PS-type SMBVis preferably between 20 and 200 nm.

[0041] The crosslinked hydrophilic core can be composed of natural orsynthetic polymers which are naturally or chemically crosslinked. Use isin particular made of polysaccharides or oligosaccharides, such asstarch, dextran, cellulose and their derivatives.

[0042] Advantageously, an active principle is encapsulated in thePS-type SMBV mainly at the center of the matrix; the external part ofthe core is virtually devoid of active principle, which makes itpossible to avoid the aggregation phenomena which generally occur forparticles of small size.

[0043] One of the subjects of the invention is therefore a particulatevector composed of a crosslinked polysaccharide matrix containing anactive principle, the active principle preferably being localized mainlyat the center of the matrix.

[0044] According to yet another aspect, a subject of the invention is aprocess for charging which makes it possible to encapsulate the activeprinciple in a complete SMBV, a PS-type SMBV or an L-type SMBV, whichcan be in the form of a suspension.

[0045] The charging is carried out on the particulate vector. In orderto do this, the hydrophilic core must contain ionic groups. The processthus requires the following steps:

[0046] a) a crosslinked hydrophilic core is prepared in which ionicgroups are fixed,

[0047] b) the active principle is charged within the vector at a pHsuitable for the active principle and while supplying energy,

[0048] c) having incorporated the active principle, the vector isrecovered.

[0049] In the case of PS-type SMBV, it is difficult to use conventionalcharging methods for incorporating ionic active principles. It is truethat methods with topological control make it possible to overcome thisproblem. However, topological control requires precise adjustment of thepH which must be compatible with the active principle and the vector.

[0050] The present inventors have, therefore, developed an alternativemethod to overcome these problems. The ionic ligands grafted into thepolysaccharide network of the vectors result in a significant affinityfor the ionic active principles of opposite charge. However, thisaffinity, during the incorporation, must be controlled in order to avoidaggregation of the vectors and to make it possible to localize theactive principle mainly within the particles. For the precharging, thiscontrol requires precise adjustment of the pH or a low level ofincorporation. This aggregation is mostly due to localization of theactive principle at the surface, which localization is itself due to thepresence of ligands at the surface of the particles.

[0051] In order to effect this new type of charging, three factors comeinto play:

[0052] a) a significant affinity of the active principle for the vectorin order to provide for incorporation of the active principle: thisaffinity is created by acidic or basic ionic ligands which are graftedinto the crosslinked polymer; the density and the strength of theligands can be adjusted according to the active principle,

[0053] b) a significant dispersion of the vectors during theincorporation in order to avoid the interactions between particles whichpromote aggregation: this dispersion can be provided for by the dilutionof the vectors in the reaction medium at a concentration which issufficient to decrease the interparticulate interactions but also at aconcentration which is compatible with pharmaceutical applications,

[0054] c) the use of any means for promoting entry of the activeprinciple within the vector: the contribution of energy, in the form,for example, of stirring or of heat, will accelerate the kinetics ofentry of the active principle but will also promote dispersion of thevectors; the appropriate form of the active principle, which must besufficiently ionic to make it possible to attach the active principlebut also the least charged, in order to avoid surface interactions.

[0055] For SMBV or L-type or PS-type SMBV, it is possible to useincorporation protocols corresponding to these requirements. Thepresence of grafted ionic ligands in the crosslinked polymer providesfor attachment of the active principles for the three species.Dispersion of the vectors can be carried out by suspending PS-type SMBVin water. SMBV or L-type SMBV are prepared from acylated orpolysaccharide cores and from phospholipids dispersed beforehand inaqueous medium and are thus suspended in water. The contribution ofenergy, for example, in the form of stirring or of heat, does not damagethe SMBV. It is possible to vary the pHs and to define pH ranges whichare compatible with this type of charging.

[0056] This new process makes it possible to prepare SMBV of any typewhich are charged with active principle, while retaining the size of thebase vectors. This process thus has many advantages. This methodcomprises preparing the blank vectors, without active principle, beforethe incorporation. This makes it possible to process the blank vectorsaccording to conditions which are suitable for the vectors and which donot depend on the active principle to be encapsulated. The vectors aresubsequently charged. These conditions can, therefore, be more or lessdrastic. They also make it possible to be able to characterize the blankvector as a base entity.

[0057] Incorporating the active principle in the final step of theprocess results in the active principle, which is capable of being toxicand expensive, being handled during only one step of the process. Thisprocess thus reduces the handlings and the possible losses of the activeprinciple. It, therefore, makes it possible to be more certain asregards safety, but also more profitable.

[0058] In addition, for some active principles, the incorporationconditions can be relatively simple, which makes it possible to envisioncharging the vectors with the active principle at the time of use. Thismethod of preparation at the time of use can eliminate the problems ofstorage in the liquid state.

[0059] This new method of charging is based on the significant affinitybetween the vectors and the ionic active principles, but also on thesimple control of the incorporation by the dispersion of the vectors andthe ionic form of the active principle. It has very worthwhileadvantages: preparation of the blank vector independent of the activeprinciple, handling of the active principle in a single step and thepossibility of preparation at the time of use.

[0060] The particulate vectors according to the invention preferablyhave a diameter of between 10 nm and 5 μm and more preferably between 20and 70 nm.

[0061] These particulate vectors are intended to carry or to present attheir surface one or a number of molecules possessing biologicalactivity. Mention must be made, among these molecules, without this listbeing limiting, of:

[0062] antibiotics and antivirals,

[0063] proteins, proteoglycans, peptides,

[0064] polysaccharides, lipopolysaccharides,

[0065] antibodies,

[0066] antigens,

[0067] insecticides and fungicides,

[0068] compounds which act on the cardiovascular system,

[0069] anticancers,

[0070] antimalarials,

[0071] antiasthmatics,

[0072] compounds having an effect on the skin,

[0073] constituents of dairy fat globules.

[0074] In the examples below, a description will be given of thecharging of various products according to their characteristics, and inparticular:

[0075] a hydrophilic product of small size intended for systemicadministration,

[0076] an active principle possessing anticancer activity,

[0077] two enzymes possessing antibacterial activity, lactoperoxidaseand glucose oxidase, and

[0078] a plant extract composed of procyanidol oligomers possessing anantioxidant activity,

[0079] constituents of the fat globule of milk.

[0080] The present invention, therefore, provides a pharmaceuticalcomposition, comprising a particulate vector according to the inventionand a pharmaceutically acceptable support for its administration. Thevectors according to the invention are in particular useful fortherapeutic and immunological applications.

[0081] The present invention also provides a cosmetological compositioncomprising a particulate vector as described above, and cosmetologicallyacceptable excipients.

[0082] Finally, food compositions comprising particulate vectorsaccording to the invention form parts of the invention.

[0083] The examples which follow are intended to illustrate theinvention without limiting the scope thereof.

EXAMPLE 1 Preparation of Polysaccharide Particles with a Mean Diameterof 20 Nanometers, by Twofold Crosslinking of Dextran by PhosphorusOxychloride

[0084] 100 g of dextran (Roquette) are introduced into a 3 literjacketed reactor and are dissolved in 350 ml of demineralized water and100 ml of 1ON sodium hydroxide.

[0085] After homogenization, 35.3 ml of POCl₃ and 225 ml of 1ON sodiumhydroxide are added simultaneously.

[0086] After the end of the addition of the reactants, the reactionmixture is stirred for a further 15 minutes and then neutralized byaddition of hydrochloric acid.

[0087] The gel is diluted in 2 liters of demineralized water andhomogenized at 900 bars using a high pressure homogenizer (Westfalia).This step makes it possible to obtain matrices with a mean diameter of20.

[0088] The matrices are then washed by precipitation with ethanol inorder to remove the salts and then dried by lyophilization at aconcentration of 30 g/l of matrices and 20 g/l of ammonium bicarbonate.75 g of lyophilized matrices are recovered (reaction yield 75%).

EXAMPLE 2 Preparation of Polysaccharide Matrices Grafted by CationicQuaternary Ammonium Groups

[0089] 200 grams of amylopectin (Roquette, Lille, Fr.) are dispersed in500 milliliters of 2N sodium hydroxide in a 5 liter reactor. When thesolution is well homogenized, 93.6 grams of glycidyltrimethylammoniumchloride (Fluka, CH), corresponding to 0.5 equivalents/ glucose residue,dissolved in 150 milliliters of water, and 11.4 grams (i.e. 9.7milliliters) of epichloroydrin (Fluka, CH), corresponding to 0.1equivalents/glucose residue, are simultaneously introduced. The mixtureis homogenized for 1 to 2 hours and then left standing for 8 hours. Thepolymerized starch preparation is then brought to pH 6 by addition ofacetic acid. The gel obtained is then washed a number of times withdistilled water until all the salts and reaction by-products have beenremoved. After lyophilization, 244 grams of crosslinked gel areobtained, i.e. a reaction yield of 80%.

EXAMPLE 3 Preparation of Polysaccharide Matrices Grafted by AnionicGroups of Carboxymethyl Type (“CM-Type”)

[0090] 200 grams of dextran (Roquette) are dissolved in 300 millilitersof 7N sodium hydroxide in a 5 liter reactor. When the solution is wellhomogenized, 9.6 milliliters of epichloroydrin (Fluka, CH),corresponding to 0.1 equivalents/glucose residue, and 117.2 grams ofchloroacetic acid, dissolved in 80 milliliters of water, aresimultaneously introduced.

[0091] After stirring for 1 hour, 9.6 milliliters of epichlorohydrin and150 milliliters of 2N sodium hydroxide are added while stirringvigorously. After the end of the addition, the preparation ishomogenized for 6 hours and then left standing overnight. The gel thusobtained is suspended in 1 liter of water and acidified to pH 3-4 byaddition of 2N hydrochloric acid. The gel is then filtered and washedwith distilled water. After lyophilization, 276 grams of gel ofcarboxymethyl type are obtained, i.e. a yield greater than 80%.

EXAMPLE 4 Preparation of Hydrophilic Matrices, with a Mean Diameter of 1μm, by Crosslinking of Starch by Phosphorus Oxychloride

[0092] 100 g of wheat starch (Roquette) are introduced into a 3 literjacketed reactor and dissolved in 375 ml of distilled water and 100 mlof 10N sodium hydroxide.

[0093] The mixture is stirred for 15 minutes at room temperature.

[0094] Once the mixture is homogenized, 11 ml of POCl₃ and 50 ml of 10Nsodium hydroxide are simultaneously added. After the end of the additionof the reactants, the reaction mixture is stirred for a further 15minutes and then neutralized to pH 7 by addition of acetic acid.

[0095] The gel is washed in a centrifuge (Rousselet) for 30 minutes withdistilled water so as to remove the excess salts and reactionby-products.

[0096] The gel thus obtained is then homogenized at high pressure (500bars, Westfalia minilab homogenizer). This step makes it possible toobtain matrices with a mean size of 1 μm. The titration of 1 g ofcrosslinked gel using an automatic titrimeter (Methrom 682titroprocessor) reveals a degree of crosslinking of 0.3 meq ofphosphodiester functional groups per gram of crosslinked gel.

[0097] PS-type SMBV with a diameter of 1 μm are thus obtained.

EXAMPLE 5 Production of Ionic Polysaccharide Particles of 200 Nanometers

[0098] 15 grams of gel obtained according to Example 2, or of CM-typegel obtained according to Example 3, are dispersed in 500 milliliters ofdistilled water and homogenized by means of a Rannie MiniLab 12-51homogenizer (APV Rannie, Copenhagen, Dk). The homogenization pressureapplied is 600 bars for 12 minutes.

[0099] A fluid suspension of basic or acidic crosslinked polysaccharideparticles is obtained, the size of the particles, measured with aCoulter N4MD Nanosizer, being centered around 200 manometers. Thenanoparticles are then dried by lyophilization in the presence of 20grams/liter of ammonium bicarbonate.

[0100] PS-type SMBV with a diameter of 200 nm are thus obtained, whichcan be used as is or converted to L-type SMBV.

EXAMPLE 6 Preparation of Anionic Polysaccharide Cores (PSC)

[0101] 500 g of maltodextrin (Glucidex, Roquette, Lestrem, France) arepoured in a 10 liter reactor (TRIMIX) along with 2 liters ofdemineralized water. After solubilization at 4° C., 500 ml of sodiumhydroxide (NaOH) 10M are added with mechanical stirring. When thetemperature of the solution has stabilized at 4° C., 1700 ml of 10M NaOHand 283.3 ml of POCL₃ are added under controlled flow conditions. Thecross linking reaction takes place with mechanical stirring during a 20hour period. At the end of the 20 hour period, the reacting mixture isstirred an additional 15 minutes. A volume of 5 liters of demineralizedwater is added and the pH is adjusted to 7.0 by neutralization withglacial acetic acid. The hydrogel obtained is ground underhigh-pressure. At the end of this step, the mean diameter of theparticles is approximately 60 run. Further purification proceeds asfollows:

[0102] (i) microfiltration at 0.45 μm to eliminate larger particles,(ii) diafiltration at constant volume to eliminate smaller molecules(salts, fragments of polysaccharaides, etc). The anionic polysaccharidecores (PSC) are then concentrated, added to sterile flasks, and storedat ˜20° C.

EXAMPLE 7 Preparation of Cationic Polysaccharide Cores (PSC)

[0103] 500 mg of maltodextrine (Glucidex, Roquette, Lestrem, France) aresolubilized with 0.880 liters of water at 20° C., with stirring, in athermoregulated reactor. Seven grams of NaBH₄ are added and mixed for 1hour. 220 ml of NaOH 10 M are added, followed by 30.25 ml ofepichlorydrin (Fulka). After 12 hours of reaction, 382.3 g ofglycidyltrimethylammonium chloride (Fulka) are introduced and themixture is stirred for 10 hours. The resulting gel is diluted with 8liters of demineralized water and the pH is adjusted to 7.0 byneutralization with glacial acetic acid. The hydrogel obtained is groundunder high-pressure. The pressure used is 400 bars. At the end of thisstep, the mean diameter of the particles is approximately 60 nm. Furtherpurification proceeds as follows: (i) microfiltration at 0.45 μm toeliminate larger particles, (ii) diafiltration at constant volume toeliminate smaller molecules (salts, fragments of polysaccharides). Thecationic PSC are then concentrated, sampled in sterile flasks and storedat ˜20° C.

EXAMPLE 8 Loading of ddCTP in Cationic Polysaccharidic Core (PSC)

[0104] Cationic PSC, obtained according to Example 7, is conjugated toan antiviral agent: dideoxy cytidine triphosphate (ddCTP). A watersolution of ddCTP (5 mg/ml) is slowly added to the solution of cationicPSC (5.5 mg/ml). Mixing is done at room temperature under magneticstirring. The ratio of ddCTP/PSC is 10% (weight/weight) with a finalconcentration of PSC of 5 mg/ml. The preparation is incubated 2 hours atroom temperature with magnetic stirring. Free ddCTP is separated fromPSC associated ddCTP by ultrafiltration on Amicon device (100 kDa). AllddCTP concentrations are measured by spectrophotometric assays.

[0105] The following table provides an example of the association ofddCTP with cationic PSC. A quantitative association is obtained betweenddCTP and cationic PSC (yield=98%). This can be explained by theimportant affinity of the phosphate groups carried by the activeprinciple and the cationic charge carried by the PSC. These associationscan be generalized to all nucleosidic antiviral or nucleosidicanticancer compounds under triphosphate form. Moreover, the excellentfilterability of the ddCTP/PSC clearly demonstrates the absence ofaggregation phenomena during the incorporation process. Association ofddCTP and cationic PSC PSC Free Entrapment yield % 98.2 ± 0.3 0Filtration yield %  971 ± 0.5 99.4 ± 0.8

EXAMPLE 9 Loading of hGRF in Anionic Polysaccharidic Cores (PSC)

[0106] Anionic PSC are obtained according to Example 6. hGRF (Synthetichuman growth hormone releasing factor (1-29)-NH2) in solution indistilled water (6 mg/ml ) is introduced drop by drop under ultrasonicsin the anionic PSC solution (1.1 mg/ml) (charge 1.7 mEq/g). Solution isleft 15 min under ultrasonics, then 4 hours at room temperature. hGRFassociated to PSC is separated from free hGRF by ultrafiltration onMicrosep (Filtron 300 Kda). hGRF concentration is then measured by UVspectrometry in the ultrafiltration supernatants.

[0107] In order to evaluate the incorporation stability, 0.9 ml ofPSC-hGRF is mixed with 0.1 ml of PBS* (concentrated 10 times), thenincubated 18 hours at 37°. hGRF associated to NPS is separated from freehGRF by ultrafiltration on Microsep (Filtron 300 Kda). hGRFconcentration is then measured by UV spectrometry in ultrafiltrationsupernatants.

[0108] The following table gives results obtained with a PSCconcentration of 1 mg/ml and an initial ratio hGRF/NPC of 60%. Underthese conditions, association between hGRF and PSC is quantitative withincorporation yields higher than 90%. Furthermore, as shown by thestability obtained in PBS, the association hGRF and PSC is stable inphysiologic medium. Incorporation of hGRF in PSC hGRF alone hGRF WITHPSC Average SD Average SD Incorporation yield (%) 4.9 3.5 92.6 2.5Incorporation ratio — — 55.5 4.9 hGRF/PSC (%) Loss in PBS (%) — —  6.52.1

EXAMPLE 10 Loading of Insulin in Cationic Polysaccharidic Cores (PSC)

[0109] 500 mg insulin (human recombinant insulin zinc salt ) aresolubilized in 15.5 ml HCl (0.02M), then neutralized to pH 8.0 with NaOHsolution (0.1 M). A solution of cationic PSC (22 g/l) (charge 1.8 mEq/g) obtained according to Example 7 is slowly added to the insulinsolution. The preparation obtained by this method is sterilized byfiltration on 0.2 μm filters. It can be used for nasal administrationafter introduction in a spray. Insulin associated to PSC is separatedfrom free insulin by Centricon (100 kDa ) ultrafiltration after{fraction (1/10)} dilution in PBS (1 mM Na2HPO₄/NaH₂PO₄, 12 mM NaCl &0.27 mM KCl ). Insulin concentration is then measured by HPLC inultrafiltration supernatants.

[0110] The following table gives results obtained for the preparation ofa clinical batch. With quantitative (100%) association yields, theobtained results show the excellent affinity of insulin for the cationicPSC structure. In these conditions, the obtained preparations areintroduced in monospray (Pfeiffer), allowing the delivery of 100 μl,corresponding to a therapeutic dose of 56.6 IU insulin with 2 mg of PSCfor an administration volume of 100 μl in each nostril. PSC Associationyield 100% PSC Concentration g/l 9.6 ± 0.2 Insulin concentration IU/ml285 ± 4  Delivered volume (μl) 93 ± 5 

EXAMPLE 11 Incorporation of an Enzyme, Lactoperoxidase (LP), in PS-TypeSMBV

[0111] Lactoperoxidase (LP) is an antibacterial enzyme. It is a basicprotein having an isoelectric point of 9.6 and an average molecularweight of approximately 80,000 daltons. 0.5 gram of anionic (CM) PS-typeSMBV, obtained according to Example 3 and then 5, is suspended in 100milliliters of a buffer adjusted to pH 7, below the pI of LP, in a 250ml round-bottomed flask. 0.5 g of LP (BioSerae), dissolved in 1milliliter of water, is then introduced with stirring.

[0112] The mixture is stirred overnight in a refrigerator (4° C.). ThepH is then adjusted to 9.8, above the pI of LP, and incubated for 30min. The pH is then brought back to 7 and the suspension is thenlyophilized in the presence of ammonium bicarbonate (20 grams/liter).PS-type SMBV charged with LP are obtained with a charging yield of 99%and a degree of incorporation of 99% with respect to the weight of thecores, from quantitative determination by LTV at 412 nm.

EXAMPLE 12 Antibacterial Activity of the LP Encapsulated in PS-Type SMBV

[0113] Antibacterial Activity Against a Strain of Escherichia coli:

[0114] An LB glucose culture medium, mixed with a gelose agar, isprepared and poured into antibiogram dishes. The strain of E. coli isinoculated at the surface of the gelose at the rate of 200 μl/dish.Sterile paper disks are impregnated with suspensions of encapsulated ornonencapsulated enzymes and deposited on the gelose of the inoculateddishes. The dishes are left to incubate for 24 h at 37° C. and theinhibition diameters around the disks are measured.

[0115] The disks were impregnated with enzyme concentrations varyingfrom 0.05 to 0.6 mg/ml of LP. The inhibition diameters vary from 12 to20 mm and are comparable, whether or not the enzyme has beenencapsulated.

EXAMPLE 13 Stabilization of the Antibacterial Activity of EnzymeEncapsulated in PS-Type SMBV

[0116] A 0.1 mg/ml aqueous lactoperoxidase solution and a suspension ofLP encapsulated in PS-type SMBV prepared according to Example 11 and inwhich the LP concentration is also 0.1 mg/ml is prepared.

[0117] These two suspensions are left at 4° C. and quantitativelydetermined every week and then every month by the method described inExample 12. The activity of the LP solution decreases with time and,after 90 days, the residual activity is no more than 35% of that of theinitial activity. In contrast, the activity of the suspension of LPencapsulated in PS-type SMBV stays constant and remains equal to 100% ofthe initial activity after 90 days.

EXAMPLE 14 Incorporation of an Anticancer Antibiotic, Doxorubicin, inPS-Type SMBV by Using the Method of Charging by Topological Control

[0118] Doxorubicin is an anticancer antibiotic belonging to theanthracycline family. It is an amphiphilic product characterized by apolyaromatic aglycone, conferring characteristic fluorescence propertieson the molecule, and by an amino sugar, daunosamine. The molecularweight of the hydrochloride is 580 and its pKa is 8.5.

[0119] Polysaccharide cores prepared as above are used.

[0120] 1. Incorporation of Doxorubicin Without Topological ControlDoxorubicin (0.1 g) in aqueous solution is added progressively to thepolysaccharide cores (0.5 g) with magnetic stirring. The suspensionobtained is then left stirring for 17 h at room temperature and with thelight excluded.

[0121] The polysaccharide cores thus charged with doxorubicin havecompletely precipitated. Even in the presence of detergent and ofphospholipids, they cannot be correctly dispersed with a size of 20 nm.

[0122] The incorporation of doxorubicin without topological controlleads to a complete aggregation of the polysaccharide cores and cannotbe used for forming L-type SMBV of 20 nm.

[0123] 2. Incorporation of Doxorubicin With Topological ControlDoxorubicin (0.1 g) in aqueous solution is added progressively to thepolysaccharide cores (0.5 g) with magnetic stirring. The pH is adjustedto 7, below the pKa of doxorubicin, during the addition. The suspensionobtained is stirred for 17 h at room temperature and with the lightexcluded. The pH is then adjusted to 9, above the pKa of doxorubicin,and incubated for 30 min.

[0124] After the incubation step at pH 9, the suspension obtained isdiluted in 1 l of water and brought to pH 7. The cores thus charged areanalyzed: filtration through 0.2 μm of the suspension exhibits a yieldof doxorubicin of greater than 95%, which indicates that the size of thepolysaccharide cores is 20 nm, and centrifugal ultrafiltration of analiquot of the suspension demonstrates the absence of free doxorubicin.The results indicate the presence of 4 mg of doxorubicin in theultrafiltrate and of 46 mg of doxorubicin in the polysaccharide cores,which corresponds to a yield of 92% and a degree of encapsulation of 18%of doxorubicin. Filtration through 0.2 gm of the suspension obtainedexhibits a yield of greater than 95%, which indicates that the size ofthe SMBVs is 20 nm.

EXAMPLE 15 Comparison Between the Incorporation of Doxorubicin inPS-Type SMBV and SMBV

[0125] Doxorubicin is an anticancer antibiotic belonging to theanthracycline family. It is an amphiphilic product characterized by apolyaromatic aglycone, conferring characteristic fluorescence propertieson the molecule, and by an amino sugar, daunosamine. The molecularweight of the hydrochloride is 580 and its pKa is 8.2-8.5.

[0126] Incorporation of Doxorubicin in Polysaccharide Cores

[0127] The polysaccharide cores used were crosslinked and functionalizedby POCl₃ and have a size of 20 nm. Their ionic density is 1.59 mequivPO4/g.

[0128] The polysaccharide cores (10 mg) are dispersed in water (10 ml)under ultrasound. The pH of the cores suspension is adjusted to 7 with0.1N NaOH. The doxorubicin (4.6 mg), as a 5 mg/ml solution in water, isslowly added while sonicating in 20 μl portions. The pH is adjusted to7, if necessary, with 0.1N NAOH.

[0129] The polysaccharide cores thus charged are characterized by theirability to be filtered through 0.2 μm. The filtration yield isdetermined by the ratio of the concentrations before and afterfiltration. After incorporation, 100 μl of the suspension of chargedpolysaccharide cores are withdrawn in order to determine the doxorubicinconcentration. The remainder of the suspension is filtered through amembrane with a porosity of 0.2 μm. A 100 μl aliquot is again withdrawnfor the quantitative determination of the doxorubicin. The doxorubicinis quantitatively determined by HPLC after release of the polysaccharidecores. $\begin{matrix}{{Filtration}\quad {yield}} \\{{through}\quad 0.2\quad {{\mu m}(\%)}}\end{matrix} = {\frac{\begin{matrix}{{doxorubicin}\quad {concentration}} \\{{after}\quad {filtration}}\end{matrix}}{\begin{matrix}{{doxorubicin}\quad {concentration}} \\{{before}\quad {filtration}}\end{matrix}} \times 100}$

[0130] the nonincorporated fraction which is determined by centrifugalultrafiltration. After filtration, 1 ml of the suspension ofpolysaccharide cores, diluted to ½, is deposited on the centrifugalultrafiltration system (Microsep) and then centrifuged at 7500 g for 30min. The ultrafiltrate obtained is quantitatively determined by HPLC fordoxorubicin. $\begin{matrix}{{Nonincorporated}\quad} \\{{fraction}\quad (\%)}\end{matrix} = {\frac{\begin{matrix}{{doxorubicin}\quad {concentration}} \\{{in}\quad {the}\quad {ultrafiltrate}}\end{matrix}}{\begin{matrix}{{doxorubicin}\quad {concentration}} \\{{after}\quad {filtration}}\end{matrix}} \times 100}$

[0131] The filtration yield is 97% and the nonincorporated fraction isless than 5%, leading to an incorporation yield of 99%.

[0132] Comparison of the Behavior Under Physiological Conditions ofDoxorubicin Incorporated in Polysaccharide Cores and in SMBV

[0133] The particles which are postcharged in doxorubicin,polysaccharide cores or SMBV, are incubated in PBS at 37° C. at adoxorubicin concentration of 50 μg/ml. At time 0 h and 4 h, 1 ml of theparticle suspension is withdrawn and ultrafiltered by centrifuging (7500g, 30 min) on a Microsep in order to determine the doxorubicin fractionreleased. The ultrafiltrate obtained is then quantitatively determinedfor doxorubicin by HPLC. The results are presented in the followingtable: % of doxorubicin remaining Type of particles Type of particlesincorporated PS-Type SMBV SMBV time 0 h 67 +/− 1 62 +/− 1 time 4 h 64+/− 3 55 +/− 5

Behavior Under Physiological Conditions of Polysaccharide Cores and ofSMBV which Have Incorporated Doxorubicin by Postcharging

[0134] The results obtained indicate a difference in behavior ofdoxorubicin incorporated in these two types of particles: doxorubicinremains incorporated more strongly in the polysaccharide cores than inthe SMBV.

[0135] The descriptions of the foregoing embodiments of the inventionhave been presented for purposes of illustration and description. Theyare not intended to be exhaustive or to limit the invention to theprecise form disclosed, and obviously many modifications and variationsare possible in light of the above teachings. The embodiments werechosen and described in order to best explain the principles of theinvention and its practical application to enable thereby others skilledin the art to best utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of the invention be defined by the claimsappended hereto. All references cited herein are incorporated byreference.

1. A synthetic particulate vector comprising a non-liquid hydrophiliccore, wherein said vector does not have an external lipid layer graftedthereon.
 2. The synthetic particulate vector according to claim 1,wherein the hydrophilic core comprises a matrix of polysaccharides oroligosaccharides which are naturally or chemically crosslinked.
 3. Thesynthetic particulate vector according to claim 1, wherein ionic ligandsare grafted onto the hydrophilic core.
 4. The synthetic particulatevector according to claim 1, further comprising an active principle. 5.The synthetic particulate vector according to claim 4, wherein theactive principle is an ionizable molecule localized mainly at the centerof the matrix.
 6. The synthetic particulate vector according to claim 1,wherein said vector has a diameter of between 10 nm and 5 μm.
 7. Thesynthetic particulate vector according to claim 6, wherein said vectorhas a diameter of between 20 and 200 nm.
 8. The synthetic particulatevector according to claim 4, wherein said active principle is selectedfrom the group consisting of antibiotics, antiviral agents, proteins,proteoglycans, peptides, polysaccharides, lipopolysaccharides,antibodies, antigens, insecticides, fungicides, compounds which act onthe cardiovascular system, anticancer agents, antimalarial agents,antiasthmatic agents, compounds having an effect on the skin, andconstituents of dairy fat globules.
 9. A process for preparing aparticulate vector according to claim 4, wherein the vector is chargedwith an active principle, said process comprising: (a) preparing theparticulate vector comprising a non-liquid hydrophilic matrix on whichare fixed ionic ligands; (b) suspending the particulate vector in asolution at a pH at which the active principle is weakly ionized; (c)adding the active principle to the suspension of (b) while supplyingenergy; (d) recovering from the solution the particulate vector which ischarged with active principle.
 10. A process for preparing a particulatevector according to claim 4, said method comprising: (a) encapsulatingan acidic or basic ionizable active principle in a crosslinkedhydrophilic matrix grafted by ligands of an ionic species of oppositeionic charge with that of the active principle, at a pH at which theactive principle is in the ionized form; (b) varying the pH of themedium, with respect to the pK_(a) of the active principle, to a valueat which the active principle is not in an ionized form; and (c)recovering the hydrophilic matrix which comprises the active principlelocalized mainly at the center of said matrix.
 11. The process forpreparing a particulate vector according to claim 10, comprising: (a)encapsulating a basic ionizable active principle in a crosslinkedhydrophilic matrix grafted by acidic ionic ligands, at a pH below thepK_(a) of the active principle; and (b) increasing the pH of the mediumto a value above the pK_(a) of the active principle.
 12. The process forpreparing a particulate vector according to claim 10, comprising: (a)encapsulating an acidic ionizable active principle in a crosslinkedhydrophilic matrix grafted by basic ionic ligands, at a pH above thepK_(a) of the active principle; and (b) decreasing the pH of the mediumto a value below the pK_(a) of the active principle.
 13. The process forpreparing a particulate vector according to claim 10, wherein thehydrophilic matrix comprises polysaccharides or oligosaccharides whichare naturally or chemically crosslinked.
 14. A pharmaceuticalcomposition of matter comprising a particulate vector according to claim1, and a pharmaceutically acceptable support for administration thereof.15. A cosmetological composition comprising a particulate vectoraccording to claim 1, and cosmetologically acceptable excipientstherefor.
 16. A process for preparing a pharmaceutical compositioncomprising encapsulating an acidic or basic ionizable active principlein a particulate vector according to claim
 1. 17. A method of treating amedical condition comprising administering a vector according to claim 1to a patient in need of such treatment.
 18. A synthetic particulatevector consisting essentially of a non-liquid hydrophilic matrix. 19.The synthetic particulate vector according to claim 18, wherein thehydrophilic core comprises a matrix of polysaccharides oroligosaccharides which are naturally or chemically crosslinked.
 20. Thesynthetic particulate vector according to claim 18, wherein ionicligands are grafted onto the hydrophilic core.
 21. The syntheticparticulate vector according to claim 18, further comprising an activeprinciple.
 22. The synthetic particulate vector according to claim 21,wherein the active principle is an ionizable molecule localized mainlyat the center of the matrix.
 23. The synthetic particulate vectoraccording to claim 18, wherein said vector has a diameter of between 10nm and 5 μm.
 24. The synthetic particulate vector according to claim 23,wherein said vector has a diameter of between 20 and 200 nm.
 25. Thesynthetic particulate vector according to claim 21, wherein said activeprinciple is selected from the group consisting of antibiotics,antiviral agents, proteins, proteoglycans, peptides, polysaccharides,lipopolysaccharides, antibodies, antigens, insecticides, fungicides,compounds which act on the cardiovascular system, anticancer agents,antimalarial agents, antiasthmatic agents, compounds having an effect onthe skin, and constituents of dairy fat globules.